A Novel Alternaria Species Isolated from Peucedanum japonicum in Korea.

We isolated and examined a new Alternaria sp., which causes leaf spots on Peucedanum japonicum in Korea, by using molecular and morphological methods. Phylogenetic analysis based on a combined internal transcribed spacer region analysis and two protein-coding genes (gpd and Alt a1) demonstrated that the causal fungus was most closely related to A. cinerariae and A. sonchi, and relevant to A. brassicae. However, conidial morphology indicated that it is a novel species within the genus Alternaria, and therefore we have assigned the fungus a new name in this study.

Alternaria is a ubiquitous fungal genus associated with a wide variety of substrates including seeds, plants, agricultural products, animals, soil, and the atmosphere. Most species are well-known and significant plant pathogens causing a range of diseases and post-harvest rots of numerous agronomic and ornamental plants [1]. Some species are commonly found as saprophytes in soil and decaying plant tissues, or as endophytes in various plants [2, 3]. In addition, several taxa have emerged as animal or human pathogens [4]. Alternaria species have been generally identified and classified based on cultural and conidial morphology [5, 6, 7]. Phylogenetic analyses of multiple gene loci have also been used to identify Alternaria species [8, 9, 10], which revealed eight nine lineages (sections) (aternantherae, alternaria, brassicicola, gypsophilae, panax, porri, radicina, and sonchi) and one species group (infectoria) [10]. Recently, Alternaria has been redefined with its allied genera based on sequence analysis of six gene regions [11], which demonstrated that the Alternaria clade is comprised of 24 lineages (sections) and six monotypic lineages. In the complex clade, 13 other genera have been reported as synonymies of Alternaria.

Peucedanum japonicum Thunb. is a medicinal plant belonging to the family Apiaceae and is widely distributed in southern and eastern Asia, including Korea [12]. The root is used in the treatment of cough and headache in Korea [13], and the leaf is a medicinal herb used for treating cough in Japan [14]. Young leaves and stems are also used as vegetables in Korea. During our studies on the genus Alternaria in Korea, a fungus believed to be an Alternaria sp. based on its morphological characteristics, was repeatedly isolated from P. japonicum leaf spot lesions [5]. We aimed to identify the Alternaria sp. that we isolated from P. japonicum by using morphological and molecular methods.

MATERIALS AND METHODS

Isolates

The fungus-infected leaves of P. japonicum were collected from Yesan, Chungnam, Korea, in August 2011. Leaf spots were round or irregular, pale brown to dark brown with a pale gray spot in the center, and sometimes surrounded with yellow halos (Fig. 1A). Segmented leaf lesions were placed in Petri dishes with moist filter paper and incubated at 25℃ to induce sporulation. Single spores were collected using a sterile glass needle and transferred onto potato dextrose agar (PDA; Difco, Montreal, Canada) plates to establish pure cultures (CNU 111485). In addition, the Alternaria isolate CNU 3010 (Jinan, Korea; September 1999), derived from P. japonicum and isolated by Yu [5], was used for comparison. Isolates were deposited in the Culture Collection of Chungnam National University (CNU) in Daejeon, Korea, and the Korean Agricultural Culture Collection (KACC) in Suwon, Korea.

Fig. 1

Symptomatology and morphology of the present fungus. A, Symptoms on Peucedanum japonicum leaves; B, Obverse (left) and reverse (right) of colony on potato dextrose agar at 25℃ for 7 days; C, Conidiophores and conidia produced on potato carrot agar at 22℃ for 7 days (scale bar: C = 50 µm).

DNA extraction and PCR amplification

Isolates were grown on PDA for 5~7 days. Mycelia were collected from the growing colonies on PDA plates for DNA extraction. Genomic DNA was extracted following a previously described method [15] with some modifications. The internal transcribed spacer (ITS) region with the primers ITS5 and ITS4 [16], glyceraldehyde-3-phosphate dehydrogenase (gpd) gene with the primers gpd1 and gpd2 [17], and Alternaria allergen a 1 (Alt a1) gene with the primers Alt-a1-for and Alt-a1-rev [9] were amplified in a 50 µL reaction mixture using Taq DNA polymerase (Solgent, Daejeon, Korea) in a GeneAmp PCR System 2700 thermo cycler (Applied Biosystems, Carlsbad, CA, USA). The resultant products were purified using a Wizard PCR prep kit (Promega, Madison, WI, USA) and sequenced with FS DyeTerminator using an ABI automated DNA sequencer. The obtained sequences (ITS, gpd, and Alt a1) were deposited in GenBank with the accession numbers KF728230, KF728242, and KF889362 for CNU 3010 and KF728231, KF889361, and KF889363 for CNU 111485.

Phylogenetic analyses

The obtained and relevant sequences available in the GenBank database (Table 1) were aligned using CLUSTAL_X ver. 2.0.11, and were adjusted manually [18]. The combined dataset of ITS, gpd, and Alt a1 gene sequences was analyzed using RAxML software [19]. Maximum likelihood analysis with 1,000 bootstrap replicates was performed using the GTRGAT model of nucleotide substitution. Stemphylium botryosum Wallroth ATCC 42170 was used as an outgroup for phylogeny analysis.

Table 1

Alternaria isolates used for phylogenetic analyses in this study

Morphological observations

The isolate CNU 111485 was used for subsequent morphological descriptions. Colony characteristics (color, size, and texture) were assessed after 7 days of growth on the PDA plates at 25℃ in the dark. Conidial morphology was examined under standard conditions as per Simmons [7]. Isolates were inoculated on potato carrot agar (PCA; 20 g white potato, 20 g carrot, and 20 g agar in 1 L). The plates were stored in a chamber without humidity control (a gradually drying atmosphere in unsealed plates) at 22℃ under a fluorescent light/dark cycle of 8/16 hr for 7 days. Conidia were mounted in lactophenol picric acid solution (Fluka, Washington, USA) and measured using an OLYMPUS BX50 light microscope (Olympus, Tokyo, Japan) with an Artcam 300MI digital camera (ARTRAY, Tokyo, Japan). Randomly selected conidia (50) were counted for morphological descriptions.

RESULTS

The combined ITS, gpd, and Alt a1 datasets resulted in an alignment containing a total of 1,538 characters including alignment gaps, which comprised 524, 564, and 450 characters of the three genes, respectively. Maximum likelihood analysis was used to develop the phylogenetic tree shown in Fig. 2. The two isolates CNU 111485 and CNU 3010 had identical sequences in each of the three genes, and formed a strongly supported clade (100% bootstrap values) in the phylogram. The two isolates were most closely related to A. cinerariae Hori & Enjoji and A. sonchi Davis, although the relationship was supported by low bootstrap values (< 70%). The two isolates were also relevant to A. brassicae (Berkeley) Saccardo, as they fell in a well-supported clade (96%).

Fig. 2

Phylogenetic tree obtained from maximum likelihood analysis of combined internal transcribed spacer, gpd, and Alt a1 gene sequences from present isolates and relevant species. Bootstrap values (≥ 70%) based on 1,000 replicates are shown above branches. The bar indicates nucleotide substitutions per site. Lineages were referenced from Woudenberg et al. [11].

The cultural and morphological characteristics of the two present isolates CNU 111485 and CNU 3010 were consistent. Conidial morphology differed from any known Alternaria species, including those of the phylogenetically relevant species A. cinerariae, A. sonchi, and A. brassicae (Table 2). Therefore, the present fungus described here is a novel species, Alternaria peucedani S. H. Yu, sp. nov.

Table 2

Morphological characteristics of present isolates and relevant species

aConidia produced on potato carrot agar at 22℃.

bConidia produced on V8 juice agar at 22℃.

cConidia produced on V8 juice agar at 20℃.

Taxonomy

Alternaria peucedani S. H. Yu, sp. nov. (Fig. 1).

MycoBank

MB805939

Description

Colonies on PDA (Fig. 1B) were well developed, 35~40 mm in diameter after 7 days incubation at 25℃ in the dark, mycelium immersed and partly superficial, effuse, cottony, obverse olivaceous buff to olivaceous, reverse olivaceous buff to dark olivaceous. Conidiophores (Fig. 1C) arose singly, laterally, or terminally from hyphae, were pale brown, straight, or slightly curved, sometimes slightly swollen at the apex, commonly only one pigmented terminal conidiogenous site, smooth-walled with 1~7 transverse septa, 25~100 (~135) µm long, 4.5~9 (~10) µm wide. Conidia (Fig. 1C) on PCA were solitary, obclavate, smooth or roughed, pale brown to dark brown, 50~120 (~127) × 15~50 (~59) µm in size, 3~11 transverse septa, 2~9 longitudinal septa, mature conidia containing excessive swelling cells, constricted at eusepta. Conidial beaks were blunt-tapered, pale brown, 15~40 (~47) µm long with 0~2 septa, 3.5~6 (~8) µm wide.

Etymology

Peucedani refers to the genus of the host plant.

Holotype

Korea, Chungnam, Yesan, from Peucedanum japonicum Thunb. Leaf, August 2011, by S. H. Yu and J. X. Deng, CNUMH 11023 (dried cultures), ex-type cultures deposited in CNU (CNU 111485) and also in KACC.

DISCUSSION

The fungus we isolated from the leaf spots of P. japonicum was characterized based on phylogenetic assessment and morphological characteristics. The results indicated that the fungus is a novel species belonging to the genus Alternaria, consistent with a previous report by Yu [5], who considered it a new Alternaria taxon based on morphological characteristics without prior naming. Here, we propose a new name, Alternaria peucedani S. H. Yu, sp. nov., to accommodate the species. To determine the pathogenicity of the fungus to P. japonicum, a conidial suspension (1 × 105 conidia/mL) was dropped onto surface-sterilized leaves. Disease spots were seen after two days, which spread quickly and within days of inoculation. The results indicate that the species is the causal agent of leaf spots disease in P. japonicum (data not shown).

Fourteen selected species representing nine phylogenetic species groups of Alternaria [10] were analyzed with the A. peucedani isolates. The resultant phylogram generated from the three-gene combined dataset (ITS, gpd, and Alt a1) exhibited similar topology as reported previously (Fig. 2) [8, 9, 10]. Recently, the nine species groups have been shown as 10 lineages (nine sections and one monotypic lineage) among the 30 lineages in the Alternaria complex clade, and some section names were changed by Woudenberg et al. [11]. Phylogenetic analysis revealed that A. peucedani is closely related to A. cinerariae and A. sonchi in the sect Sochi, and comparative to A. brassicae, a monotypic lineage. However, A. peucedani is distinguishable from both the Sochi sect and the monotypic lineage (Fig. 2).

Based on conidial morphology, A. peucedani is most similar to A. cinerariae. Their mature conidia are characterized by cell hypertrophy with no predictable patterns and strongly inflated conidia. A similar situation is also seen in A. panax Whetzel [7], which is phylogenetically distant from A. peucedani and A. cinerariae (Fig. 2). A. peucedani conidia are solitary in culture, as shown by Yu [5] and in the present study, while those of relevant species (A. cinerariae, A. sonchi, and A. brassicae) are often catenulate with 2~3 conidia [7]. Additionally, the conidial body of A. peucedani is wider and shorter than that of A. brassicae and A. cinerariae, but larger than that of A. sonchi. A. peucedani also produces chlamydospores (12~15 µm wide) on V8 juice agar [5], but this structure is not observed on PCA.